Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR) were isolated from two human genomic DNA fragment libraries. One clone (lambda hDHFR-1) contains three coding blocks (exons) which encode the C-terminal half of human DHFR. The other two groups of recombinant each contain an intronless DHFR coding sequence. One recombinant (lambda h DHFR-psi2) includes the entire coding sequence; the presence of several in phase termination codons in all three reading frames indicate that DHFR-psi2 is a pseudogene. The overall sequence homology of hDHFR-psi2 to the available portions of the normal human DHFR coding sequence is 93%. On the other hand, the other intronless gene, (hDHFR-psi1) has an open reading frame and is virtually identical to the coding sequence of the normal gene with introns. The DNA sequence homology among the 3 genes extends 2.9 kilobases beyond the end of the coding sequences. Both intronless genes have an A-rich tract at the 3' end of the sequence homology. The lack of introns and the presence of A-rich tracts at the 3' ends of intronless genes suggest that these genes are derived from processed RNA intermediates. A short DNA sequence, 60 nucleotides 5' to the ATG initiation codon in hDHFR-psi2 is directly repeated immediately after the 3'A-rich tract providing evidence of DNA insertion.